Enhancement of cellular gallium uptake

ABSTRACT

A method for improving cellular gallium uptake (particularly of tumor cells) by exposing cells to a nifedipine photodegradation product, or an analog thereof. In particular embodiments, the gallium uptake enhancer is selected from the group of A-B and formula (I), wherein A is a pyridine and B is a nitrosophenyl, and n=1-10. In yet other embodiments, the uptake enhancer is formula (II), wherein R 1-9  are independently selected from the group consisting of H, halogen, haloalkyl, NO 2 , NO, SO 2 , a C1-6 alkyl, a COOR 10  wherein R 10  is H or C1-6 alkyl, and an —OR 11  wherein R 11  is H or C1-6 alkyl; wherein at least one of R 5  and R 7  is NO. The uptake enhancers are particularly useful in imaging tumors, using such techniques as gallium scanning, in which the dose of the gallium isotope can be decreased or its imaging efficiency improved. Alternatively, the method can be used to improve efficacy of gallium containing chemotherapeutic regiments in the treatment of tumors and hypercalcemia, or to improve the uptake of other chemotherapeutics that use a similar transferrin independent uptake mechanism.

PRIORITY

This patent was filed under 35 U.S.C. § 371 and claims priority fromPatent Cooperation Treaty (PCT) International Patent ApplicationPCT/US99/07879, filed Apr. 8, 1999, which in turn claims priority fromU.S. Provisional Patent Application 60/081,336, filed Apr. 8, 1998. Bothpriority applications are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to increasing the uptake of gallium into cellsfor diagnostic and therapeutic purposes.

BACKGROUND OF THE INVENTION

Gallium (Ga), a Group IIIa transition metal, has a number of isotopeswith many medical uses. For decades, gallium-67, a gamma-emitter, hasbeen used in nuclear medicine for tumor imaging by gamma emissionscintigraphy (1). Currently, gallium-67 is most widely used in stagingand assessing the therapeutic response of lymphomas (2, 3, 4, 5). Otherisotopes of gallium have potential uses in oncology. Gallium-68, apositron emitter, can be used for tumor imaging by positron emissiontomography (PET). Gallium-72, a beta-emitter, may destroy tissues thatconcentrate gallium by local radiation. This treatment has been proposedto palliate bone pain caused by skeletal metastases (6). Gallium-67 hasalso been used for local radiotherapy in the treatment of hematologicalmalignancies (48, 49, 50, 51).

Stable (non-radioactive) gallium has been used to reduce thehypercalcemia of malignancy, and as a treatment for Paget's disease ofbone. It is also believed to have direct anti-neoplastic effects, and iscurrently under investigation as an adjunct to conventional chemotherapy(7, 8, 9).

The limitations of Ga-67 for oncologic imaging are well-recognized(10,11,12,13). Many tumors accumulate Ga poorly. Others, such ashepatomas and lymphomas, can be intensely Ga-avid but may vary inmagnitude and consistency of uptake. Delineation of tumors frombackground tissues often requires extended intervals from the time ofinjection to imaging of 3-7 days or more because Ga-67 localizes slowlyand initial images of the abdomen are frequently difficult to interpretbecause of bowel activity. Because of the extended intervals requiredfor oncologic imaging, a relatively high dose of Ga-67 is required(typically 10 mCi for an adult). Despite its drawbacks, no othergamma-emitting radiopharmaceutical used for tumor imaging in nuclearmedicine (including expensive monoclonal antibodies and receptor-avidpeptides) has surpassed Ga-67 in cost-effectiveness, generalavailability, broad applicability and ease of imaging. Although effortsto improve the use of gallium are clearly justifiable, the techniques toaccomplish this have thus far been elusive for impractical.

Despite years of imaging experience with the Ga-67 radiometal, themechanism by which Ga-67 accumulates in normal tissues and tumorsremains controversial. For years, it has been thought that gallium istaken up by cells as a gallium-transferrin (Ga-Tf) complex via thetransferrin receptor (TfR) (14,15,16). However, there is also evidencethat mechanisms other than the TfR may be responsible for the uptake ofGa-67 in tumors (17,18,19). For example, gallium may dissociate from Tfin the acidic extracellular environment of tumors, which would interferewith Tf mediated transport of cellular uptake (20, 21, 22). There isalso a poor correlation between TfR density and the degree of tumoruptake of gallium. Moreover, gallium uptake continues to a significantdegree even in the absence of Tf, or when TfR binding sites are blockedwith an antibody or when iron overload down regulates TfR expression(23, 24, 25).

Tumor bearing rats that are rendered iron-deficient (which increasesTfR's in many tissues) exhibit an increased uptake of Ga-67 in tissueother than tumors (26). When Tf binding sites are saturated with iron orscandium after administration of Ga-67, uptake of gallium in tumors,relative to normal tissues, can actually increase (27, 28). Uptake ofGa-67 by nonosseous tissues and organs is markedly depressed in ahypotransferrinemic strain of mouse, suggesting that uptake of Ga-67 bymost soft tissues and organs is a Tf-dependent process (29).

Nifedipine 1 (dimethyl1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylate) isa dihydropyridine calcium channel antagonist, which causes vasodilationand lowering of peripheral vascular resistance. These characteristicsmake nifedipine useful in the treatment of heart disease andhypertension. This compound, like most1,4-dihydro-4-(2-nitrophenyl)pyridine derivatives, is very sensitive tolight. Photo-degradation of nifedipine has been considered a drawback toits pharmaceutical use, because the photo-degradation products have beenthought to lack pharmacological activity. Hence photo-degradation ofnifedipine has diligently been avoided by shielding it from the light toprevent loss of its therapeutic properties.

In the presence of light, nifedipine is converted to phenylpyridinederivative structures that include fully-aromatic compounds (FIG. 1).With exposure to visible/fluorescent light, nifedipine is convertedpredominantly to the 4-(2-nitrosophenyl)pyridine homologue 2 (thenitroso derivative, also known as2,6-dimethyl-3,5-diacetyl-4-(2′-nitrosophenyl)-pyridine). When exposedto UV light, it is converted predominantly to the4-(2-nitrophenyl)pyridine homologue 3 (the nitro nifedipine derivative).The nitro derivative is also the primary metabolic product of nifedipinein humans. In addition to these two main structures, photo-degradednifedipine (PDN) also includes a broad variety of phenylpyridines suchas the cis and trans-azoxy derivatives, the hydroxylamine derivative,the amine derivative, the lactam derivative, and the trans-N,N′-dioxidederivative.

SUMMARY OF THE INVENTION

The present invention takes advantage of an unexpected property ofnifedipine degradation products, such as photodegraded nifedipineproducts (PDN), or pharmaceutical analogs and their degradationproducts. This property can be used to improve the use of gallium forseveral purposes: 1) to improve tumor imaging; 2) to improveradiotherapy of tumors; and 3) to improve the use of gallium as anadjunct to chemotherapy. In particular example, the method can improvethe uptake of gallium into tumor cells, to permit a total diagnostic ortherapeutic dose of the radioisotope to be decreased, so that less thanthe normal 5-10 mCi adult dose can be administered to an adult.

There are several mechanisms by which PDN can improve the use of galliumisotopes, such as Ga-67 (for gamma scintigraphy), for tumor imaging.First, PDN selectively augments a Tf-independent uptake of gallium, andsince tumors appear to accumulate gallium by this route to a greaterextent than normal tissues, PDN could improve the localization ofgallium selectively in tumors. Even if PDN stimulates uptake of galliumin normal tissues as well as tumors, it still has significant beneficialeffect in decreasing the necessary interval between time of injection ofthe radio-tracer and time of imaging. Improving the efficiency of uptakeof gallium in tumors or other tissues allows diagnostic images to beobtained at a lower dose of radioactivity to the patient. Tumor specificenhancement of gallium uptake by PDN improves the use of stable galliumas an adjunct to conventional chemotherapy, and concentration ofunstable gallium isotopes in tumors for the purpose of administeringlocal radiotherapy.

The present invention therefore includes exposing cells, tissues ortumors to a sufficient dose of the PDN products, for a sufficient periodof time, to improve the uptake of gallium into the cells or tumors. Thecells can be exposed to the PDN in vitro (for example is an assay) byproviding the photo-degradation products (or biological precursors) in asurrounding medium. Alternatively, the PDN can be administered to cells,tissues or tumors in vivo to achieve a systemic blood level, or a localconcentration in a tissue of interest (such as a tumor), sufficient toincrease gallium uptake in that tissue. Either the PDN productsthemselves can be administered, or a biological precursor (such asnifedipine) can be administered and allowed to degrade. The degradationmay occur by normal metabolic pathways to one of the photo-degradationproducts. However, the degradation may alternatively be induced byexposure to light, such as pre-irradiation of a solution of nifedipineprior to its administration, or use of light delivered to the tissue ofinterest (for example through external or endoscopic fiberoptic lightdelivery of the kind used in photodynamic therapy).

Nifedipine is well-absorbed orally and achieves peak plasma levelsapproximately 30 minutes post ingestion. In humans treated withnifedipine, a typical dose range is 0.5-2.0 mg/kg/day, given orally inthree equally-divided daily doses. It is anticipated that PDN will besimilarly well-tolerated and well-absorbed orally, although it may alsoprove effective if given by other routes, such as by intravenous,subcutaneous or intramuscular injection. PDN is likely to be effectivein a dose range similar to that for nifedipine to achieve a local tissueconcentration in the range of 0.25-25 μM. Even higher tissueconcentrations can be used, because the PDNs are relatively otherwisepharmacologically inert. In vitro, cells which are exposed to the PDNcompounds in this concentration range for as little as 10 seconds showenhanced gallium uptake.

Any number of the individual PDN structures, such as those shown in FIG.1, may demonstrate activity in promoting gallium uptake. Theseparticular PDN products can include nitroso-nifedipine,dehydro-nifedipine, the cis or trans-azoxy nifedipine derivative, thetrans-N,N′-dioxide nifedipine derivative, the hydroxylamine, amine orlactam derivatives, or any other degradation products of nifedipine orother dihydropyridine that increases the uptake of gallium into cells.The cells which are exposed to these compounds are, for example, tumorcells. However, the method of the present invention can also be usedwith other cells or tissues in vivo in which concentration of gallium isincreased by exposure to nifedipine photo-degradation products.

The invention also includes pharmaceutical compositions of nifedipinephoto-degradation products or their precursors, either in isolation orin combination with a pharmaceutical carrier, and in unit dosage forms.All routes of administration of PDN products or their precursors areincluded in this invention. The invention also includes methods ofdiagnosis and treatment in which nifedipine (or another4-phenyldihydropyridine derivative) is intentionally exposed to light(such as visible or ultraviolet light) to produce the photo-degradationproducts. This intentional exposure can take place either prior toadministration of the drug to a subject, or in situ in the body. Theperiod of exposure of the nifedipine to light is for a sufficient periodof time to produce an adequate concentration of photo-degradationproducts, for example at least about 1 minute, or 1 to 5 minutes, oreven several hours, for example about 4 hours, or as long as a day ormore. This invention also includes pharmaceutical compositions ofphoto-derivatives of nifedipine that are used to promote gallium uptake,regardless of whether these derivatives are produced byphoto-irradiation or by alternate methods, such as chemical synthesis.

In particular embodiments, the invention includes a method of increasinggallium uptake by a cell, by exposing the cell to an effective amount ofa gallium uptake enhancer comprising a nifedipine photodegradationproduct, or an analog thereof, that promotes gallium uptake by the cell.The cells are (simultaneously or substantially concurrently) exposed toa gallium compound such as a salt containing a stable or unstableisotope. The gallium compound may be, for example, gallium nitrate,gallium citrate or gallium chloride. Examples of the gallium metal orisotope are Ga-67, Ga-68, GA-69, Ga-71 and Ga-72 (where Ga-69 and 71 arestable isotopes, and the others are unstable radioactive isotopes).

The cell which is exposed to the gallium and the uptake enhancer may bea tumor cell, so that uptake of chemotherapeutic amounts of gallium intothe tumor can be differentially increased, compared to non-tumor cells.The cells can also substantially simultaneously be exposed to adjuvantchemotherapeutic anti-neoplastic pharmaceutical agents, such asvinblastine, ifosfamide, hydroxyurea, paclitaxel, cisplatin,methotrexate, 1-beta-D-arabinofuranosylcytosine, and etoposide.Particular examples of tumor cells which could be exposed to the galliumand uptake enhancer are a sarcoma, myeloma, renal adenocarcinoma,testicular leydig cell tumor, medullary thyroid carcinoma,neuroblastoma, melanoma, colon adenocarcinoma, lung adenocarcinoma, orintraductal breast carcinoma.

In yet other embodiments, the method is used to increase uptake ofgallium into bone, for example to treat bone specific conditions such asosteoporosis, or to treat hypercalcemia (such as hypercalcemia caused byhyperparathyroidism or malignancy), or to treat Paget's disease of bone.

The disclosed methods can be used to increase cellular gallium uptakeeither in vitro or in vivo. For in vivo applications, the gallium andthe gallium uptake enhancer are administered to a subject, such assomeone who has been diagnosed with a tumor. The gallium may beadministered in a therapeutically effective antineoplastic amount, whencombined with the gallium uptake enhancer. Alternatively, the galliummay be administered in an amount effective to image the tumor in agallium scan, when the gallium is administered in combination with thegallium uptake enhancer. Combined administration does not requiresimultaneous administration, but can refer to simultaneous,substantially simultaneous or separate administration. In particularembodiments, the gallium uptake enhancer is administered prior to thegallium, but within a sufficient period of time to enhance uptake by thetissue of interest (such as the tumor).

Disclosed embodiments of the invention include a gallium uptake enhancerwhich enhances gallium uptake by a transferrin independent mechanism.Particular examples of such enhancers include a nitrosophenylpyridine,such as the 2′-nitrosophenyl photodegradation product of nifedipine, ora 2′- or 4′-analog thereof. The 2′-nitroso-nifedipine photodegradationproduct (labeled “nitroso-derivative” in FIG. 1) is believed to beparticularly effective in promoting gallium uptake.

In yet other embodiments, the gallium uptake enhancer is selected fromthe group consisting of:

A-B and

wherein A is a pyridine and B is a nitrosophenyl (such as a2′-nitrosophenyl or 4′-nitrosophenyl), and n=1-10. Alternatively, thegallium uptake enhancer is:

wherein

R₁₋₄, R₆, and R₈₋₉ are independently selected from the group consistingof H, halogen (particularly Cl), haloalkyl (particularly CCl₂), NO₂, NO,SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁wherein R₁₁ is H or C1-6 alkyl;

and R₅ and R₇ are independently selected from the group consisting of H,halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ isH or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl, wherein atleast one of R₅ and R₇ is NO.

In particular embodiments in which one of R₅ and R₇ is NO, R₁₋₉ areselected from the group of H, a C1-6 alkyl, and COOR₁₀, where R₁₀ islower alkyl, such as methyl or ethyl. In some embodiments, R1═R2═loweralkyl such as methyl, and R4═R5═an ester, such as COOCH₃. R₅ may be NO,and R₆₋₉═H.

In particular embodiments, R₁₋₄, R₆, and R₈₋₉ are independently selectedfrom the group consisting of C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H orC1-6 alkyl, and —OR₁₁ wherein R₁₁ is H or C1-6 alkyl. In even moreparticular embodiments, R₅ is NO and R₇ is H; R₁═R₂═H, R₃═R₄═COOCH₃; andR₆═R₈═R₉═H.

Even more broadly, the gallium uptake enhancer may be selected from

wherein n=1-10, and

wherein R₁₋₄, R₆, and R₈₋₉ are independently selected from the groupconsisting of H, halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, aCOOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H orC1-6 alkyl;

and R₅ and R₇ are independently selected from the group consisting of H,halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ isH or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl, wherein atleast one of R₅ and R₇ is NO.

or wherein

R₁₋₄, R₆, and R₈₋₉ are independently selected from the group consistingof H, NO, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an—OR₁₁ wherein R₁₁ is H or C1-6 alkyl;

and R₅ and R₇ are independently selected from the group consisting of H,NO, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁wherein R₁₁ is H or C1-6 alkyl, wherein at least one of R₅ and R₇ is NO,particularly R₅.

Particular embodiments of the method include imaging a tumor with agallium scan, by administering to a subject an effective amount of agallium uptake enhancer, such as a nifedipine photodegradation product,or an analog thereof, that increases uptake of gallium by a tumor. Asufficient amount of gallium is also administered to the subject toperform the gallium scan, wherein the sufficient amount of gallium isless than required to perform the gallium scan in the absence of thegallium uptake enhancer. When the gallium uptake enhancer is atransferrin independent gallium uptake enhancer such as a2′-nitrosophenylpyridine, transferrin independent uptake selectivelyconcentrates the gallium in the tumor to improve the imaging signalobtained from the tumor. When the method is used to improve imaging oftumors, Ga-67 is a particularly suitable isotope, and 50% or less of theusual dose of 10 millicuries of gallium can be administered to performthe scan. Hence a dose of less than about 5 millicuries of the Ga-67 canbe used. The uptake enhancer can also allow the tumor to be imaged muchmore quickly than in the absence of the enhancer. Hence instead ofwaiting 36-72 hours to obtain the image, the diagnostic procedure can beperformed 24 hours or less after administration of the gallium.

In embodiments in which a nifedipine photodegradation product (such as2′-nitrosophenylpyridine derivative) is administered to the subject, adose of about 0.5 to about 2.0 mg/kg/day of the nifedipinephotodegradation product may be employed. However, the nifedipinephotodegradation products are not known to have any biological effect(other than enhancing gallium uptake). In particular, they do not act ascalcium channel antagonists. Hence even much higher doses of nifedipinephotodegradation products can be used.

In yet other embodiments in which a cutaneous tumor (such as a melanoma)is to be treated, the gallium uptake enhancer is nifedipine applied toskin in an area of the cutaneous tumor, which area is subsequentlyirradiated with light (such as visible/fluorescent light) that producesthe nifedipine photodegradation product gallium uptake enhancer.However, cutaneous and other types of tumors may also be sensitized byadministering the gallium uptake enhancer systemically (for exampleintravenously or orally) to a subject having the tumor.

The invention also includes methods of screening for a gallium uptakeenhancer, by exposing cells to a test agent such as a nifedipinephotodegradation product, or an analog thereof, in the presence ofgallium. The uptake of gallium in the cell is then measured to determinewhether the cellular uptake of gallium is greater or less than in theabsence of the test agent. In particular disclosed embodiments, thecells are cultured Chinese Hamster Ovary (CHO) cells, such astransferrin receptor negative CHO cells.

Additional objects and advantages of the present invention will beapparent from the following detailed description of a preferredembodiment, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing of the degradation pathway of nifedipine, showingexamples of nifedipine's many intermediate degradation products. Thisinvention includes, but is not limited to, any of these illustratedstructures that enhance gallium uptake, but also includes any chemicalor photo-derivative of nifedipine or other dihydropyridines that proveseffective in improving the cellular or tissue uptake of gallium.

FIG. 2 shows the structural formula of the dihydropyridine calciumchannel blocker, nimodipine.

FIG. 3 shows the structural formula of the dihydropyridine calciumchannel agonist, BAY K 8644.

FIG. 4 is a chart illustrating gallium uptake by tumor cells. A solutionof 25 mM nifedipine was exposed to a strong fluorescent light source for4 hours. The photo-degraded nifedipine was then incubated with tumorcells at a concentration of 25 μM for 30 minutes in the presence ofGa-67 citrate (PDN Ga-67 uptake). Control tumor cells were incubated inthe absence of PDN in the presence of Ga-67 citrate for 30 minutes(Ga-67 uptake).

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

Abbreviations

Tf: Transferrin

TfR: Transferrin receptor

TfR−: Transferrin receptor negative (lacking a transferrin receptor)

TfR+: Transferrin receptor positive (having a transferrin receptor)

PDN: Photo-degraded nifedipine

Definitions

The following definitions will help with an understanding of the termsused in this specification.

A “gallium uptake enhancer” is an agent that increases the amount ofgallium in a cell above the amount that is present in the absence ofsuch a gallium uptake enhancer.

A “transferrin-independent gallium uptake enhancer” is a gallium uptakeenhancer that acts by, but is not limited to, a mechanism independent oftransferrin. A transferrin-independent gallium uptake enhancer may alsoincrease gallium uptake by a mechanism dependent on transferrin.

“Gallium” includes isotopes of gallium, such as Ga-67, Ga-68, Ga-69,Ga-71, or Ga-72, (where Ga-69 and 71 are stable, and Ga-67, 68, 70 and72 are unstable), and compounds such as gallium nitrate, galliumcitrate, or gallium chloride salts.

A “gallium scan” is a nuclear medicine imaging technique in which aradioactive isotope of gallium, such as Ga-67, is given to a patientintravenously. After administration, the gamma emissions are measuredwith a gamma camera which produces a photographic image that correlatesintensity of tissue uptake with darkness of image. The photographicimage provides information that is useful for diagnosis and therapeuticassessment.

A “PET scan” is a nuclear medicine imaging technique in which aradioactive isotope of gallium that emits positrons, such as Ga-68, isadministered to a patient intraveneously. After administration, thepositron emissions are measured and the information is used fordiagnosis and therapeutic assessment.

“Visible light” includes light having a wavelength between about 380-760nm. “Ultraviolet light” has a wavelength immediately below visible(violet) light, and extends from about 100-380 nm.

A “tumor” is a neoplasm, and includes both benign and malignant tumors.This term particularly includes malignant tumors which can be eithersolid (such as a breast, liver, or prostate carcinoma) or non-solid(such as a leukemia). Tumors can also be further divided into subtypes,such as adenocarcinomas (e.g. of the breast, prostate or lung).

A “therapeutically effective dose” is a dose sufficient to preventadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease.

“Fully-aromatic ring system” is a ring system (such as a phenylpyridine)in which both rings of the system are aromatic.

The term “alkyl” refers to a cyclic, branched, or straight chain alkylgroup containing only carbon and hydrogen, and unless otherwisementioned contains one to twelve carbon atoms. This term is furtherexemplified by groups such as methyl, ethyl, n-propyl, isobutyl,t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Alkylgroups can either be unsubstituted or substituted with one or moresubstituents, e.g. halogen, alkyl, alkoxy, alkylthio, trifluoromethyl,acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryloxy, aryl, arylalkyl,heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino,pyrrolidin-1-yl, piperazin-1-yl, or other functionality.

The term “lower alkyl” refers to a cyclic, branched or straight chainmonovalent alkyl radical of one to six carbon atoms, but can alsoinclude up to 3, 4 or 5 carbon atoms. This term is further exemplifiedby such radicals as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl,i-butyl (or 2-methylpropyl), cyclopropylmethyl, i-amyl, and n-amyl.Lower alkyl groups can also be unsubstituted or substituted, where aspecific example of a substituted alkyl is 1,1-dimethyl propyl.

“Hydroxyl” refers to —OH.

“Carboxyl” refers to the radical —COOH, and includes both unsubstitutedand substituted carboxyl. “Substituted carboxyl” refers to —COR where Ris alkyl, lower alkyl or a carboxylic acid or ester.

The term “aryl” refers to a monovalent unsaturated aromatic carbocyclicgroup having a single ring (e.g. phenyl) or multiple condensed rings(e.g. naphthyl or anthryl), which can optionally be unsubstituted orsubstituted with, e.g., halogen, alkoxy, mercapto (—SH), alkylthio,trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl,arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino,piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.

The term “alkoxy” refers to a substituted or unsubstituted alkoxy, wherean alkoxy has the structure —O—R, where R is substituted orunsubstituted alkyl. In an unsubstituted alkoxy, the R is anunsubstituted alkyl. The term “substituted alkoxy” refers to a grouphaving the structure —O—R, where R is alkyl which is substituted with anon-interfering substituent.

The term “heterocycle” refers to a monovalent saturated, unsaturated, oraromatic carbocyclic group having a single ring (e.g. benzyl,morpholino, pyridyl or furyl) or multiple condensed rings (e.g.naphthyl, quinolinyl, indolizinyl or benzo[b]thienyl) and having atleast one heteroatom, defined as N, O, P, or S, within the ring, whichcan optionally be unsubstituted or substituted with, e.g. halogen,alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto,carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino,dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl,or other functionality.

The term “halogen” refers to fluoro, bromo, chloro and iodosubstituents.

A “pharmaceutical agent” or “drug” refers to a chemical compound orcomposition capable of inducing a desired therapeutic or prophylacticeffect when properly administered to a subject.

All chemical compounds include both the (+) and (−) stereoisomers, aswell as either the (+) or (−) stereoisomer.

An analog is a molecule, that differs in chemical structure from aparent compound, for example a homolog (differing by an increment in thechemical structure, such as a difference in the length of an alkylchain), a molecular fragment, a structure that differs by one or morefunctional groups, or a change in ionization. Structural analogs areoften found using quantitative structure activity relationships (QSAR),with techniques such as those disclosed in Remington: The Science andPractice of Pharmacology, 19^(th) Edition (1995), chapter 28. Aderivative is a biologically active molecule derived from the basestructure.

Other chemistry terms herein are used according to conventional usage inthe art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms(1985) and The Condensed Chemical Dictionary (1981).

A “mammal” includes both human and non-human mammals. Similarly, theterm “subject” includes both human and veterinary subjects.

An animal is a living multicellular vertebrate organism, a categorywhich includes, for example, mammals and birds.

The following Examples show that the photo-degradation products of thepresent method improve gallium uptake in cultured cells, and areintended to illustrate, but not limit, embodiments of the presentinvention.

EXAMPLE 1 Cell Line and Culture

A pair of transfected Chinese Hamster Ovary cells lines were used tocompare, in a controlled manner, the Tf-dependent and Tf-independentsystems for the uptake of Ga-67. Details regarding plasmid construction,and the transfection, selection, and characterization of the cells havebeen recently described (37), and that disclosure is incorporated byreference.

The two cell lines are identical except that TfR-cells express no TfR,and TfR+ cells over-express the transfected human TfR constitutively.This means that expression of the TfR is independent of cell growth oriron content, which could alter the cells matabolically in many waysthat may confound a well-controlled experimental determination of causeand effect. TfR− and TfR+ cells were grown in monolayer and maintainedas previously described (37).

EXAMPLE 2 Photo-degradation of Nifedipine

Nifedipine (Sigma Chemical Co., St. Louis Mo.) was dissolved in 1 mlethanol at a concentration of 10 mM. Care was taken to shield nifedipinefrom the light except during intentional irradiation. Forphoto-irradiation by fluorescent light, the nifedipine in ethanol wasplaced in a clear 10 ml polystyrene conical bottom screw cap tube. Thetube was placed on its side on the surface of a cool, daylightcolor-balanced, fluorescent light box (Just Normlicht). For irradiationby UV light, the nifedipine solution was placed in a quartz glasscuvette and placed on the surface of a UV light box (Fotodyne) in anotherwise dark cabinet for 4 hours. The interval of photo-irradiationranged from 1 minute to 24 hours.

The temperature at the surface of the light box was 29° C. for thefluorescent light box, and 37° C. for the UV light box. The PDN wasadded to 10 ml of incubation solution (below) to achieve concentrationsranging from 0.25 μM to 100 μM. Each 10 ml sample of incubation solutioncontained an equal quantity of ethanol (approximately 100 □l per 10 ml),including the controls.

EXAMPLE 3 Gallium Uptake

Following growth of cells in 25 cm² flasks to subconfluence, monolayersof cultured cells were first washed, and then pre-incubated for 2 hoursat 37° C. with 5 ml serum-free Dulbecco's Modified Essential Medium(DMEM) to deplete the cells of Tf. The pre-incubation medium was thenremoved and replaced by 1.5 ml of the incubation solution containing 10uCi/ml carrier-free Ga-67 citrate (Mallinckrodt) in Hank's Balanced Saltsolution (HBSS), pre-warmed to 37° C. The concentration of Ga-67 in theincubation solution was approximately 0.25 nM. The HBSS, pH 7.2-7.4,contained 3.7 g/L NaHCO3, 1 mM CaCl₂, and 1 mM magnesium salts. Cellswere incubated in the PDN- and Ga-67-containing solutions in a CO₂incubator at 37° C. for intervals ranging from 10 seconds to 90 minutes.All experiments were conducted in the dark.

Following incubation of cells with Ga-67, the flasks were immediatelyplaced on ice. The radioactive material was removed by aspiration with aPasteur pipette attached to water suction. The monolayers were washed 3times with 5 ml each ice cold HBSS. Cells were washed once withphosphate-buffered saline (PBS), pre-warmed to 37° C. The PBS wasremoved and the cellular monolayer overlaid with 1.5 ml 0.25% trypsincontaining 1 mM EDTA. The trypsin was immediately removed and the cellswere incubated briefly (1 minute) at 37° C. The cells in each flask werethen dislodged by several gentle mechanical blows to the side of theflask and collected in 200 μl ice cold PBS.

By a modification of a method previously published for radiolabeling ofprotozoan parasites (38), the cells were then separated from unboundcontaminating radioactivity. The 200 μl of cells in PBS were layeredgently over 200 μl of an 8.5:1.5 ratio of dibutylpthalate:liquidparaffin oil in a 1.5 ml microfuge tube. With care not to agitate themixture, the tubes were than centrifuged at 12,000 rpm for 2 minutes ina microfuge. The supernatant and the oil was then aspirated carefullyfrom the top of the tube with a Pasteur pipette attached to watersuction. The bottom of the microfuge tube, containing the cell pellet(typical pellet volume˜100 μl), was then clipped with a microfuge tubeclipper into a counting vial containing 900 μl of a solution of 200 mMNaOH. 1% SDS. The cell pellet was dissolved in this solution. Thecontent of radioactivity in the samples was determined by a gamma wellcounting (Packard) in comparison to standard dilutions of the originalGa-67 incubation solution.

Protein assays were performed on the solubilized cell samples byformation of a cuprous bicinchonicic acid complex using aspectrophotometric microtiter plate reader (Dynatech) (39). The methodand reagents used are supplied in a kit (Pierce) and were performedaccording to the manufacturer's directions.

By photo-irradiation for intervals ranging from 1 minute to 24 hours,the time required for maximal conversion of nifedipine to a form thatwould promote uptake of Ga-67 was determined (Table 1, below).Nifedipine shielded from the light has no effect on uptake of Ga-67.Exposure to as little as 1 minute of UV or fluorescent light results ina product that stimulates Ga-67 uptake 2-3 fold over basal levels. Thesame maximal degree of Ga-67 uptake is produced by nifedipine irradiatedby UV as by fluorescent light, approximately 1000-fold greater thanbasal levels. However, there were some differences between the UV andfluorescent effects. Maximal uptake of Ga-67 is produced by 4 hours offluorescent irradiation of nifedipine, while irradiation for only 1 hourof UV light is required for the maximal effect. There is no loss ofactivity for nifedipine continuously exposed to fluorescent light for 24hours. However, continuous exposure of nifedipine to UV light forintervals longer than 1 hour results in progressive diminution ofactivity.

TABLE 1 Effect of Length of Photo-Irradiation of Nifedipine on CellularUptake of Ga-67 Time of fmoles Ga-67/mg Photo-irradiation Total CellularProtein (SEM) of Nifedipine TfR− cells TfR+ cells no nifedipine 0.200(0.130) 0.243 (0.009) protected from light .171 (0.011) 0.296 (0.013) 1min 0.572 (0.105) 0.644 (0.093) 5 min 30.339 (2.206) 25.479 (2.457) 1 h141.605 (9.769) 139.607 (9.710) 4 h 185.097 (9.913) 201.249 (9.914) 24 h182.199 (12.036) 196.803 (7.181)

A solution of 10 mM nifedipine was exposed to a strong fluorescent lightsource for various lengths of time shown at left. The photo-degradednifedipine was then incubated with cultured TfR− and TfR+ cells at aconcentration of 25 μM for 30 minutes in the presence of Ga-67 citrate.

Stimulation of uptake of Ga-67 by nifedipine irradiated by either UV orfluorescent light is a concentration-dependent phenomenon. Concentrationof PDN as low as 0.25 μM result in an uptake of Ga-67 that is 40-foldgreater than control levels. Maximal uptake of Ga-67, approximately1000-fold greater than control levels, is achieved by 25 μM of eitherthe UV or fluorescent-irradiated nifedipine (Table 2). With higherconcentrations of nifedipine, no further increase in uptake wasobserved. With the UV-irradiated product, there is actually a slight butsignificant decline in activity when the concentration is raised from 25to 100 μM. Basal and stimulated levels of uptake of Ga-67 are notaltered by either pre-incubation of the cells with 100 μM light-shieldednifedipine, or by it's addition to the incubation mixture containing the25 μM of PDN.

TABLE 2 Effect of Concentration of Photodegraded Nifedipine on CellularUptake of Ga-67 fmoles Ga-67/mg Concentration of Total Cellular Protein(SEM) Photo-Degraded Nifedipine TfR− cells TfR+ cells 0 μM 0.186 (0.15)0.210 (0.018) 0.25 μM 8.218 (0.855) 7.525 (1.113) 5 μM 39.250 (5.514)36.608 (3.793) 25 μM 210.513 (8.255) 208.101 (13.599) 100 μM 210.365(19.276) 222.554 (11.909) A solution of 25 μM nifedipine was exposed toa strong fluorescent light source for 4 hours The photo degradednifedipine was then incubated at various concentrations with culturedTfR− and TfR+ cells for 30 minutes in the presence of Ga-67 citrate.

Cellular uptake of Ga-67 in the presence of nifedipine degraded byeither UV or fluorescent light is very rapid, and does not requirepre-incubation with PDN. With even 10 seconds of incubation with Ga-67in the presence of PDN, uptake of Ga-67 is 6-10 fold greater than basallevels (achieved by cells incubated with Ga-67 alone for 30 minutes).With 30 minutes of exposure to Ga-67 and photo-degraded nifedipine,uptake of Ga-67 is 1000-fold greater than basal levels (Table 3, below).

TABLE 3 Effect of Time of Incubation with Photo-Degraded Nifedipine onCellular Uptake of Ga-67 fmoles Ga-67/mg Time of Incubation with TotalCellular Protein (SEM) Photo-Degraded Nifedipine TfR− cells TfR+ cellscontrol 0.186 (0.015) 0.210 (0.018) (no nifed.) 30 min  10 sec 1.784(0.262) 1.334 (0.378)  1 min 6.550 (0.834) 9.892 (0 989)  5 min 65.735(9.428) 73.506 (8.167)   min 125.463 (6.196) 117.397 (7.583) 30 min181.674 (5.911) 201.525 (15.837) 90 min 243.373 (11.794) 253.843 (8.534)A solution of 25 μM nifedipine was exposed to a strong fluorescent lightsource for 4 hours. The photo degraded nifedipine was then incubatedwith cultured TfR− and TfR+ cells at a concentration of 25 uM forvarious intervals in the presence of Ga-67 citrate. The control wasincubated in the absence of nifedipine for 30 minutes.

TfR+ and TfR− cells demonstrate equivalent degrees oftransferrin-independent uptake of Ga-67 and of stimulation of uptake byPDN. Therefore, the mechanism stimulated by the nifedipine derivativesis unrelated to expression of the TfR, or to any contaminatingtransferrin in the medium. The TfR− and TfR+ cells, derivatives of CHOcells, are not unique in their enhancement of Ga-67 uptake in responseto PDN. Two other lines of cultured cells (Balb/3T3 cells transformed bythe Moloney Murine Sarcoma Virus and NIH 3T3 cells, American TypeCulture Collection) have also been tested and demonstrate a pattern andmagnitude of PDN-stimulated Ga-67 uptake similar to that of theCHO-derived cells.

EXAMPLE 4 Nifedipine Photodegradation Products

The isolation, identification and kinetics of formation of thephoto-degradation products of nifedipine have been described (40,41) andsome of the known degradation products are shown in FIG. 1. As alreadynoted, these derivatives have previously been considered undesirablebecause they lack pharmacological activity. Exposure of nifedipine todaylight or fluorescent light results predominantly in thenitroso-derivative degradation product (FIG. 1), which is relativelystable. The other products are either formed only transiently or insmall amounts after extended exposure to daylight or fluorescent light(42,43). The dehydro (nitro)-derivative (FIG. 1) is the primarymetabolic product of nifedipine in humans and is also the majorphoto-degradation product resulting from ultraviolet irradiation ofnifedipine. Since irradiation by both visible and UV light result inproducts that promoted the same magnitude of uptake of Ga-67, multiplecompounds resulting from the photo-degradation of nifedipine arebelieved to be effective in promoting the uptake of Ga-67.

EXAMPLE 5 The Nitroso-Derivative of Nifedipine Increases Gallium Uptake

The data in Example 3 indicate that the nitroso-derivative (FIG. 1) isone of the photo-degradation products that enhances gallium uptake,because the maximal uptake of Ga-67 was observed under conditions thatcorrelate with the presence of the nitroso-derivative. Specifically, thenitroso-derivative predominates and is stable in fluorescent light, andthe maximal activity of gallium uptake is observed and sustained under 4hours of fluorescent irradiation of nifedipine. Moreover, thenitroso-derivative is only transiently produced by UV light (42), andcontinuous exposure of nifedipine to UV light for intervals longer than1 hour results in progressive diminution of gallium uptake activity.

EXAMPLE 6 Gallium Uptake Unexpectedly Superior to Iron Transport

Photo-degraded nifedipine has been reported to increase thetransferrin-independent uptake of Fe²⁺ in nucleated rabbit erythrocytes.The nitroso-derivative of nifedipine was isolated and had the samepharmacological action as “crude” photo-degraded nifedipine in enhancingthe uptake of Fe²⁺ in these cells (44). However, the augmentation ofuptake by photo-degraded nifedipine is much greater for Ga-67(1,000-fold) than was reported for Fe²⁺ (4-fold). Hence, gallium uptakeis about 250 times greater than the reported increase in Fe²⁺ uptake inthe presence of nifedipine degradation products.

Photo-degraded nifedipine also fails to promote the uptake of iron inthe trivalent state (44). Gallium is thought to exist in biologicalsystems only in the trivalent form (45). Whether or not gallium and ironshare the same system for transferrin-independent uptake, photo-degradednifedipine appears to act as a much more effective ionophore for galliumthan for iron.

EXAMPLE 7 Gallium Uptake Activity in Photo-degraded Structural andFunctional Analogs of Nifedipine

Using the methods explained in Examples 1-3, Ga-67 uptake in thepresence of other calcium channel blockers and other dihydropyridinesunder both light-protected and photo-irradiated conditions was measured.Two non-dihydropyridine calcium channel blockers, diltiazem andverapamil, one dihydropyridine calcium channel blocker, nimodopine (FIG.2), and one dihydropyridine calcium agonist, BAY K 8644 (FIG. 3), weretested. None of the light-protected or photo-irradiated compounds had aneffect on Ga-67 uptake. Thus, neither calcium channel activity norphoto-irradiation of a dihydropyridine is predictive of gallium uptakeactivity. Nifedipine itself, if not irradiated, is also not active.

Comparison of the structures of the active nitroso-derivative (FIG. 1)and the inactive dihydropyridines, nimodipine (FIG. 2) and BAY K 8644(FIG. 3), reveals that the position of the nitroso group and the fullyaromatic ring system (in which both the pyridine and phenyl ring arearomatic) contribute to gallium uptake activity. For instance, theactive nitroso-derivative (FIG. 1) has a nitroso group in the 2′position of the phenyl ring and a fully aromatic pyridine ring. Neithernimodipine (FIG. 2) nor BAY K 8644 (FIG. 3) has a fully aromaticphenylpyridine ring system. BAY K 8644 does not contain a nitroso group,and although nimodipine (FIG. 2) does have a nitroso group, it is in the3′ position on the phenyl ring.

Structures that are predicted to enhance gallium uptake include, but arenot limited to, structures sterically similar to the activenitroso-derivative. These structures include, but are not limited to,nitrosophenyl-pyridines with the nitroso-group in the 2′ or the 4′position of the phenyl ring, and substitutions of various alkyl, ester,and hydroxyl groups on both the pyridine and phenyl rings. Suchsubstitutions include, but are not limited to: lower alkyls; carboxylicacids and esters of the formula COOR wherein R is an H or lower alkyl;NO₂, NO, or SO₂; or hydroxyls and ethers of the formula —OR, wherein Ris an H or lower alkyl. In other embodiments, the two phenyl rings maybe joined by 1 to 10 or more carbons, for example (C═C—C)_(n), in eitherthe cis or trans position, where n is 1-10, such as 5-10, or any numberin between 1 and 10.

EXAMPLE 8 Photo-Degraded Nifedipine Increases Gallium Uptake by TumorCells

Nifedipine exposed to either visible or UV light markedly stimulates thetransferrin-independent uptake of Ga-67 in cultured human, mouse, andrat tumor cells at relatively low concentrations (52). Using the methodsdescribed in Examples 1-3, several tumor cell lines were exposed tophoto-degraded nifedipine (PDN) to measure gallium uptake. Tumor cellswere obtained from the American Type Culture Collection (Manassas, Va.).A solution of 25 mM nifedipine was exposed to a strong fluorescent lightsource for 4 hours. The photo-degraded nifedipine was then incubatedwith tumor cells at a concentration of 25 μM for 30 minutes in thepresence of Ga-67 citrate (PDN Ga-67 uptake). Control tumor cells wereincubated in the absence of PDN in the presence of Ga-67 citrate for 30minutes (Ga-67 uptake).

Mouse tumor cells tested included embryonic sarcoma (MMSV/3T3), myeloma(XS63), renal adenocarcinoma (RAG), testicular leydig cell tumor (I-10),T-cell lymphoma (RAW 8.1), and medullary thyroid carcinoma (MTC-M). Rattumor cells tested included neuroblastoma (Neuro 2-A). Human tumor cellstested included melanoma (HT-144), colon adenocarcinoma (Caco-2), lungadenocarcinoma (Calu-1), and intraductal breast carcinoma (BT-474).Results from these experiments are shown in FIG. 4. Gallium uptakeincreased in all tumor cells tested.

EXAMPLE 9 Methods of Imaging and Treatment

Taking advantage of tumors relying to a greater degree on transferrinindependent uptake of gallium than does normal tissues, thephoto-degradation products of nifedipine can offer a method forselectively increasing the uptake of gallium by tumors. This finding iscontrary to the accepted teaching that the photo-degradation products ofnifedipine have historically been thought to lack pharmacologicalactivity. However, the absence of other pharmacological activityindicates that the use of these compounds to increase gallium uptake issafe for clinical use. The absence of other pharmacological activityalso indicates that the photo-degraded products of nifedipine can bedosed similarly to nifedipine, 0.5-2.0 mg/kg/day, but even larger dosescan be used without adverse physiological consequences. Therapeuticallyeffective doses of the gallium uptake enhancers of the present inventioncan be determined by one of skill in the art, with a goal of achievingtissue concentrations that are at least as high as that achieved withthe administration of nifedipine.

The administration of nifedipine itself to subjects to increase theuptake of gallium is also included in the present invention, because thederivative produced by UV irradiation is also the primary metabolicproduct of nifedipine in humans, and the simultaneous presence of lightshielded nifedipine does not reverse the effect of photo-degradednifedipine. Using the assays described in this specification, it ispossible to isolate individual photo-derivatives, or their analogs, andassess their ability to modulate the uptake of Ga-67. The effect of anysuch compound can also be readily assessed for efficacy as an imaging oranti-tumor agent, using the techniques described in the foregoingexamples.

The method of this invention may also be used to improve tumorlocalization of other isotopes of gallium, such as Ga-68 or Ga-72 forlocal irradiation of tumors. The method of this invention may also beused to improve the uptake of stable gallium salts, such as galliumnitrate, gallium citrate, or gallium chloride, for the purpose ofreducing bone resorption or as an adjunct to conventional chemotherapy.

EXAMPLE 10 Methods of Increasing Gallium Uptake for the Treatment ofTumors

The method includes administering the gallium uptake enhancers of thepresent invention, or a combination of the gallium uptake enhancers andone or more other chemotherapeutic anti-neoplastic pharmaceuticalagents, including stable gallium, to the subject in a pharmaceuticallycompatible carrier, and in an amount effective to inhibit thedevelopment or progression of the tumor.

The vehicle in which the gallium uptake enhancers or chemotherapeuticagents are delivered can include pharmaceutically acceptablecompositions of these substances, using methods well known to those withskill in the art. Any of the common carriers, such as sterile saline orglucose solution, can be utilized with the gallium uptake enhancers andchemotherapeutic agents of the invention. Routes of administrationinclude but are not limited to oral and parenteral rountes, such asintravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic,nasal, sublingual, and transdermal.

The specific dose level and frequency of dosage for any particularsubject may be varied, and will depend upon a variety of factors,including the activity of the specific compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex, diet, mode and time of administration, rate of excretion,drug combination, and severity of the condition of the host undergoingtherapy. Determination of a specific dose can be determined by anattending physician, according to the condition of a subject, and thepurpose for which the compound is being administered.

The present invention can be used in the treatment of a variety oftumors. Examples of such tumors include ovarian cancer, carcinoma of theurothelium, bladder cancer, bone metastases, colon cancer, lung cancer,thymoma, breast cancer, and lymphoma.

For example, VIG (vinblastine, ifosfamide, gallium nitrate) can beadministered as an anti-tumor treatment (53, 54) for ovarian cancer andadvanced carcinoma of the urothelium. Vinblastine, 0.08-0.11 mg/kg, isadministered iv on days 1 and 2, ifosfamide, 0.9-1.2 g/m² iv, on days1-5 with mesna uroprotection, gallium nitrate, 225-300 mg/m²/day, as acontinuous infusion for 120 hours or days 1-5, and G-CSF. Cycles arerepeated at 21-day intervals. Gallium uptake enhancers can be added tothese regimens to improve the tumor response.

Combination therapy with paclitaxel, G-CSF (filgrastim), galliumnitrate, and calcitriol can be administered as an anti-tumor treatment(55), for example colon cancer adenocarcinoma or thymoma. Galliumnitrate, 300 mg/m²/day, is administered as a continuous iv infusion for120 hours. For the last 24 hours of gallium administration, paclitaxel,90-225 mg/m², is administered as a continuous iv infusion for 24 hours.Calcitriol, 0.5 μg/day orally is administered on days 1-7. G-CSF, 5μg/kg/day, can be added to the regimen for higher doses of paclitaxel.The cycle is repeated every 21 days. Gallium uptake enhancers can beadded to these regimens to improve the tumor response.

Gallium nitrate, 200-350 mg/m²/day continuous iv infusion for 7 days incombination with hydroxyurea, 500-1000 mg/day orally can be used as ananti-neoplastic regimen (56), for example with non-Hodgkin's lymphoma.Gallium uptake enhancers can be added to this regimen to improve thetumor response.

Another anti-tumor combination therapy comprises cisplatin, etoposide,and gallium chloride (57), for example for small cell and non-small celllung cancers. Cisplatin and etoposide are administered as a continuousiv infusion over 5 days. Gallium chloride, 400 mg/day, is given orally.The cycles can be repeated every 21 days and 6 or more cycles can begiven.

The combination therapies are of course not limited to the listsprovided in these examples, but include any composition for thetreatment of tumors.

The present invention can also be used in the treatment of a variety ofhematological malignancies by local radiotherapy (47, 48, 49, 50, 51,58). Examples of such hematological malignancies include non-Hodgkin'slymphoma, Hodgkin's lymphoma, and leukemia.

For example in the treatment of acute lymphoblastic leukemia (ALL) andacute myelogenous leukemia (AML), a radioactive isotope of gallium, suchas Ga-67 citrate, is administered at a dose of about 36-105 mCi, ivpush, for about 12 doses. Gallium uptake enhancers in accordance withthe present invention can be administered to improve the tumor responseto this therapy. Additionally, chemotherapeutic agents such ashydroxyurea, 1-beta-D-arabinofuranosylcytosine, and methotrexate can beadded to this regimen to further improve the tumor response to thistherapy (58).

Gallium uptake enhancers can be administered locally to treat cutaneoustumors. For example, nifedipine is delivered locally to the tumor andabsorded transdermally (63, 64, 65). The nifedipine is thenphoto-irradiated, allowing the production and transdermal absorption ofthe gallium uptake enhancers, and increasing the uptake of gallium bythe tumor cells.

EXAMPLE 11 Gallium Uptake Enhancers for the Imaging of Tumors

The present invention improves the uptake of gallium in tumors (such ashepatomas and lymphomas) that already exhibit good gallium uptake, andenhances gallium uptake in tumors that previously have had poor galliumuptake. The present invention enhances the uptake of isotopes of galliumto improve gamma ray emission detection (gallium scan) or positronemission detection (PET scan) (59). This uptake enhancement allows thenormal time delay between injection and imaging of Ga-67 to besubstantially reduced (for example from the normal 72 hour delay, to24-36 hours or even less). This technique also improves thetarget:background ratio of activity between tumors (or other abnormalstructures for which gallium uptake is enhanced) and the normalbackground tissues. This gallium uptake enhancers also can reduce thedose of gallium necessary to image by as much or even more than one-halfthe amount necessary to perform the gallium scan in the absence of agallium uptake enhancer.

EXAMPLE 12 Gallium Uptake Enhancers for Reducing Bone Resorption

Stable gallium, including gallium nitrate, can be used in the treatmentof bone-resorptive diseases such as Paget's disease, osteoporosis,hypercalcemia of malignancy, multiple myeloma, blastic bone metastasis,and lytic bone metastasis (60, 61, 62). Bone treated with gallium issignificantly more resistant to cell-mediated osteolysis by osteoclasts,and bone lysis induced by parathyroid hormone and tumor necrosis factor(60). Gallium uptake enhancers can be administered with gallium, such asstable gallium, for the treatment of bone-resorptive diseases.

The hypercalcemia of malignancy can be treated by administering galliumnitrate by continuous i.v. infusion at doses of 100-200 mg/m² for 5-7days to achieve normocalcemia (60). This is approximately one-third ofthe dose used as an anti-tumor agent. Gallium uptake enhancers can beadded to this regimen to reduce the dose and duration of gallium therapynecessary to achieve normocalcemia, or to increase the speed andefficacy of the therapy.

Both lytic and blastic bone metastasis (for example metastatic prostaticadenocarcinoma) can be treated by administering gallium nitrate bycontinuous iv infusion 200 mg/m² for 5 days (60). A low dose regimen of40 mg/day by subcutaneous injection, 2 weeks on/2 weeks off for 6 monthscan also be used. Gallium uptake enhancers can be added to this regimento increase the efficacy of the gallium therapy or to reduce the doseand duration of gallium therapy necessary to achieve a therapeuticeffect.

Paget's disease can be treated by administering gallium nitrate 100mg/m²/day continuous iv infusion for 5 days (61). Gallium uptakeenhancers can be added to this regimen to increase the efficacy of thegallium therapy, or to reduce the dose and duration of gallium therapynecessary to achieve a therapeutic effect.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that the illustratedembodiment is only a preferred example of the invention and should notbe taken as a limitation on the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

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We claim:
 1. A method of increasing gallium uptake by a cell, comprising: exposing the cell to an effective amount of a gallium uptake enhancer comprising a nifedipine photodegradation product, or an analog thereof, that promotes gallium uptake by the cell.
 2. The method of claim 1, further comprising exposing the cells to gallium.
 3. The method of claim 2, wherein the gallium is a gallium compound, salt, or a gallium isotope.
 4. The method of claim 3, wherein the gallium compound is selected from the group consisting of gallium nitrate, gallium citrate and gallium chloride.
 5. The method of claim 3, wherein the gallium isotope is selected from the group consisting of Ga-67, Ga-68, Ga-69, Ga-71 and Ga-72.
 6. The method of claim 2, wherein the cell is a tumor cell, and the method further comprises administering one or more chemotherapeutic anti-neoplastic pharmaceutical agents selected from the group consisting of vinblastine, ifosfamide, hydroxyurea, paclitaxel, cisplatin, methotrexate, 1-beta-D-arabinofuranosylcytosine, and etoposide.
 7. The method of claim 2, wherein the cell is a tumor cell selected from the group consisting of a sarcoma, myeloma, renal adenocarcinoma, testicular leydig cell tumor, medullary thyroid carcinoma, neuroblastoma, melanoma, colon adenocarcinoma, lung adenocarcinoma, or intraductal breast carcinoma.
 8. The method of claim 2, wherein the cell is a bone cell.
 9. The method of claim 2, wherein the cell is exposed to the gallium and the gallium uptake enhancer by administering the gallium and gallium uptake enhancer to a subject.
 10. The method of claim 9, wherein the subject has a tumor, and the gallium in administered in a therapeutically effective antineoplastic amount, when combined with the gallium uptake enhancer.
 11. The method of claim 9, wherein the subject has a tumor, and the gallium is administered in an amount effective to image the tumor in a gallium scan, when the gallium is administered in combination with the gallium uptake enhancer.
 12. The method of claim 1, wherein the gallium uptake enhancer is a transferrin independent gallium uptake enhancer.
 13. The method of claim 1, wherein the gallium uptake enhancer is a nitrosophenylpyridine.
 14. The method of claim 13, wherein the nitrosophenylpyridine is a 2′- or 4′-nitrosophenylpyridine derivative.
 15. The method of claim 13, wherein the nitrophenylpyridine is 2′-nitrosophenylpyridine derivative.
 16. The method of claim 1, wherein the gallium uptake enhancer is selected from the group consisting of: A-B and

wherein A is a pyridine and B is a nitrosophenyl, and n=1-10.
 17. The method of claim 16, wherein B is a 2′-nitrosophenyl or 4′-nitrosophenyl.
 18. The method of claim 17, wherein the gallium uptake enhancer is:

wherein R₁₋₄, R₆, and R₈₋₉ are independently selected from the group consisting of H, halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl; and R₅ and R₇ are independently selected from the group consisting of H, halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl, wherein at least one of R₅ and R₇ is NO.
 19. The method of claim 18, wherein R₁₋₄, R₆, and R₈₋₉ are independently selected from the group consisting of C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and —OR₁₁ wherein R₁₁ is H or C1-6 alkyl.
 20. The method of claim 18, wherein R₅ is NO and R₇ is H.
 21. The method of claim 20, wherein R₁═R₂═CH₃, R₃═R₄═COOCH₃.
 22. The method of claim 21, wherein R₆═R₈═R₉═H.
 23. The method of claim 1, wherein the gallium uptake enhancer is selected from the group consisting of:

wherein n=1-10, and wherein R₁₋₄, R₆, and R₈₋₉ are independently selected from the group consisting of H, halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl; and R₅ and R₇ are independently selected from the group consisting of H, halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl, wherein at least one of R₅ and R₇ is NO.
 24. A method of imaging a tumor with a gallium scan, comprising: administering an effective amount of a gallium uptake enhancer comprising a nifedipine photodegradation product, or an analog thereof, that increases uptake of gallium by a tumor; and administering a sufficient amount of gallium to perform the gallium scan, wherein the sufficient amount of gallium is less than required to perform the gallium scan in an absence of the gallium uptake enhancer.
 25. The method of claim 24, wherein the gallium uptake enhancer is a transferrin independent gallium uptake enhancer.
 26. The method of claim 25, wherein the gallium uptake enhancer is a 2′-nitrosophenylpyridine.
 27. The method of claim 24, wherein the nifedipine photodegradation product or analog is selected from the group consisting of:

wherein n=1-10, and wherein R₁₋₄, R₆, and R₈₋₉ are independently selected from the group consisting of H, halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl; and R₅ and R₇ are independently selected from the group consisting of H, halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl, wherein at least one of R₅ and R₇ is NO.
 28. The method of claim 27, wherein the nifedipine photodegradation product or analog is:


29. The method of claim 24, wherein administering the gallium comprises administering Ga-67.
 30. The method of claim 29, wherein the effective amount of gallium is less than one-half an amount of gallium required to perform the gallium scan in the absence of the gallium uptake enhancer.
 31. The method of claim 29, wherein administering the gallium comprises administering no more than about 5 millicuries of the Ga-67.
 32. The method of claim 24, wherein the tumor is selected from the group consisting of a sarcoma, myeloma, renal adenocarcinoma, testicular leydig cell tumor, medullary thyroid carcinoma, neuroblastoma, melanoma, colon adenocarcinoma, lung adenocarcinoma, or intraductal breast carcinoma.
 33. The method of claim 29, wherein administering the gallium uptake enhancer allows imaging of the tumor at 24 hours or less after administration of the gallium.
 34. The method of claim 24, wherein administering an effective amount of gallium comprises administering about 0.5 to about 2.0 mg/kg/day of the nifedipine photodegradation product.
 35. The method of claim 34, wherein the nifedipine photodegradation product is a 2′-nitrosophenylpyridine.
 36. The method of claim 10, wherein the tumor is a cutaneous tumor, and the gallium uptake enhancer is nifedipine applied to skin in an area of the cutaneous tumor, which area is subsequently irradiated with light that produces the nifedipine photodegradation product gallium uptake enhancer.
 37. The method of claim 10, wherein the gallium uptake enhancer is administered systemically to a subject having a tumor, within an effective period of time as a gallium containing chemotherapeutic agent to increase uptake of the chemotherapeutic agent into the tumor.
 38. The method of claim 37, wherein the gallium uptake enhancer is administered orally to the subject.
 39. A method of treating a cutaneous tumor, comprising: administering nifedipine to the subject; photoirradiating the cutaneous tumor with light that causes the nifedipine to form a photodegradation product that enhances gallium uptake; administering gallium to the subject.
 40. The method of claim 39, wherein the nifedipine is administered systemically.
 41. The method of claim 39, wherein the nifedipine is applied topically to the cutaneous tumor.
 42. A method of screening for a gallium uptake enhancer, comprising: exposing cells to a test agent comprising a nifedipine photodegradation product, or an analog thereof, in the presence of gallium; measuring an uptake of gallium in the cell, and determining if the uptake of gallium is greater or less than in the absence of the test agent.
 43. The method of claim 42, wherein the cells are cultured Chinese Hamster Ovary cells.
 44. The method of claim 42, wherein the cells are transferrin receptor negative cells.
 45. A gallium uptake enhancer detected by the method of claim
 42. 46. The method of claim 1, wherein the nifedipine photodegradation product is not produced by photo-irradiation.
 47. The method of claim 46, wherein the nifedipine photodegradation product is made by chemical synthesis.
 48. A method of increasing gallium uptake by a cell in a subject, comprising: administering to the subject an effective amount of a compound that increases gallium uptake, the compound comprising:

wherein R₁₋₄, R₆, and R₈₋₉ are independently selected from the group consisting of H, halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl; and R₅ and R₇ are independently selected from the group consisting of H, halogen, haloalkyl, NO₂, NO, SO₂, a C1-6 alkyl, a COOR₁₀ wherein R₁₀ is H or C1-6 alkyl, and an —OR₁₁ wherein R₁₁ is H or C1-6 alkyl, wherein at least one of R₅ and R₇ is NO.
 49. The method of claim 48, wherein the method is a method of increasing gallium uptake in a tumor cell of the subject.
 50. The method of claim 48, wherein the compound is a 2′- or 4′-nitrosophenylpyridine derivative.
 51. The method of claim 50, wherein the compound is the 2′ nitrosophenylpyridine derivative. 